PROJECT SUMMARY/ABSTRACT Parasitic nematodes infect roughly 20% of the world's population and exact an enormous toll in human illness. Most of these parasites infect their hosts as developmentally arrested infective third- stage larvae (iL3) that resume development once they enter the host. Drawing upon findings in Caenorhabditis elegans, we have shown that the parasitic nematode Strongyloides stercoralis has significantly modified insulin-like (ILS) and steroid-nuclear hormone receptor (NHR) signaling to regulate iL3 development during the infective process. In this renewal application, we propose to expand our studies of regulation of iL3 development and morphogenesis by ILS and NHR signaling in S. stercoralis, deploying our new method for CRISPR/Cas9 mutagenesis to unambiguously assess gene function and RNAseq to determine transcriptomic changes resulting from knockout of key signaling elements. Specific Aim 1 asks whether ILS regulates formation and development of L3i, and what genes in S. stercoralis are regulated by ILS downstream. To this end, we will knock out Ss-daf- 16, which encodes an ILS-regulated transcription factor and Ss-daf-2, which encodes the insulin-like receptor kinase in S. stercoralis. We will subject knockout worms to RNAseq to ascertain global transcriptional changes and specific ones involving cytochrome P450 genes that could act in biosynthesis of Ss-DAF-12 NHR ligands. Aim 2 will assign functions to insulin-like peptides (ILPs) in S. stercoralis as agonists or antagonists of ILS by expressing them from transgenes designed to alter temporal patterns of ilp transcription in the worms. Aim 2 will also determine whether Ss-DAF-12 NHR signaling feeds back to positively regulate ILS by enhancing expression of agonistic ILPs in S. stercoralis. Aim 3 will employ vivo and in vitro studies to test the hypothesis that Ss-DAF-12 NHR signaling regulates lifespan in S. stercoralis, lengthening it in free-living adults and shortening it in parasitic females. All three aims will contribute to future development of much needed new anthelmintics targeting ILS and NHR signaling in parasitic nematodes. Elucidating functional links between these pathways will enable new combination therapies that target both pathways, acting additively or synergistically to block development and accelerate death and expulsion of adult worms.